Pharmacological antagonism of the antinociceptive effects of serotonin in the rat spinal cord

European Journal of Pharmacology, 90 (1983) 349-357 Elsevier

PHARMACOLOGICAL ANTAGONISM IN THE RAT SPINAL CORD

349

OF THE ANTINOCICEPTIVE

EFFECTS OF SEROTONIN

CLAUDIA SCHMAUSS, DONNA L. HAMMOND *, JAMES W. OCHI and TONY L. YAKSH ** Departments of Neurosurgery Research and Pharmacology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, U.S.A. Received 23 August 1982, revised MS received 11 March 1983, accepted 24 March 1983

C. SCHMAUSS, D.L. HAMMOND, J.W. OCHI and T.L. YAKSH, Pharmacological antagonism of the antinociceptive effects of serotonin in the rat spinal cord, European J. Pharmacol. 90 (1983) 349-357. Serotonin creatinine sulfate (5-HT) administered via chronic~ly implanted intrathecal catheters produced a dose-dependent increase in the rat hot plate and tail flick response latencies. To characterize the nature of the spinal receptor system through which this effect is mediated, putative serotonin antagonists were co-administered with 200/xg of 5-HT. In these experiments, methysergide (12-48 nmol), 2-bromolysergic acid diethylamide (BOL 6-48 nmol) and ketanserin (12-73 nmol) produced a dose-dependent antagonism of the effects of intrathecally administered 5-HT on the tail flick; with the IC50 values being: methysergide 17.5 + 2.5 nmol; BOL 26.5 + 6.5 nmol; ketanserin 34.5 __+5.1 nmol. The effects of spiroperidol (12-44 nmol) and metergoline (6-32 nmol) were not dose-dependent, although a measurable antagonism did occur at the highest dose. In contrast to the results obtained with the tail flick test, none of the 5-HT antagonists attenuated the elevation of hot plate latency produced by 5-HT. With the exception of spiroperidol, none of these agents showed any effect on the baseline response latencies. Sacrifice and dissection of rat brain and cord at 5, 15 and 45 rain after intrathecal [L4C]5-HT revealed that significant quantities of radioactivity appeared in brain only at the longest interval, at a time when the magnitude of the analgesia was significantly diminished, indicating that these spinal drug effects were likely produced by a local action on spinal receptors. The ability of methysergide and BOL (agents with measurable 5-HT 1 binding activity) to readily antagonize the effects of intrathecal 5-HT versus the relative inactivity of spiroperidol and ketanserin, used to characterize the 5-HT 2 binding site, suggests the likely role of 5-HT 1 receptors in mediating the spinal effects of 5-HT in the tail flick, but not the hot plate test of nociception. Analgesia

Serotonin

Intrathecal

Serotonin antagonist

1. Introduction I n t r a t h e c a l a d m i n i s t r a t i o n of s e r o t o n i n ( 5 - H T ) p r o d u c e s a b e h a v i o r a l l y d e f i n e d analgesia. This a n a l g e s i a has been shown in p r e l i m i n a r y studies to be d o s e - d e p e n d e n t a n d to be a t t e n u a t e d b y a d m i n i s t r a t i o n of p e r i p h e r a l 5 - H T a n t a g o n i s t s such as m e t h y s e r g i d e ( Y a k s h a n d Wilson, 1979). I o n t o phoresis o f 5 - H T in the spinal cord has been * Present address: Searle Research and Development Division, G.D. Searle and Company, 4901 Searle Parkway, Skokie, IL 60077, U.S.A. ** To whom all correspondence should be addressed: Mayo Clinic, Rochester, MN 55905, U.S.A.

r e p o r t e d to depress the firing of dorsal h o r n neurons which are responsive to n o x i o u s stimuli ( R a n d i c a n d Yu, 1976; H e a d l e y et al., 1978; J o r d a n et al., 1978). This i n h i b i t i o n of nocisponsive dorsal h o r n neurons m o s t likely underlies the p r o d u c t i o n o f analgesia b y i n t r a t h e c a l l y a d m i n i s t e r e d 5-HT. In other regions of the CNS, i o n t o p h o r e t i c app l i c a t i o n of 5 - H T has been r e p o r t e d to p r o d u c e either an i n h i b i t i o n or an excitation of neurons ( H a i g l e r a n d A g h a j a n i a n , 1974; 1977). Peripheral 5 - H T a n t a g o n i s t s a p p e a r to be m o r e effective in a n t a g o n i z i n g the e x c i t a t o r y effect of 5 - H T (Haigler a n d A g h a j a n i a n , 1974; M c C a l l a n d A g h a j a n i a n , 1980) than its i n h i b i t o r y effect ( H a i g l e r a n d

350 Aghajanian, 1974; Segal, 1976; Griersmith and Duggan, 1980). Such a differential effect of the peripheral 5-HT antagonists is of interest since the production of analgesia by 5-HT in the spinal cord may involve both an inhibitory action on dorsal horn neurons (postsynaptic inhibition) as well as an excitatory action on primary afferent terminals (presynaptic inhibition). These two different electrophysiological effects have been hypothesized to be mediated by two different 5-HT receptors, 5HT] and 5-HT2, respectively (Peroutka and Snyder, 1979; 1981; Hamon et al., 1980). Thus, the purpose of this study was to further characterize the pharmacological profile of the analgesia produced by intrathecally administered 5-HT with an emphasis on the type of 5-HT receptor which may mediate this effect.

2. Materials and methods

2.1. Animal preparation Male Sprague-Dawley rats weighing between 250 and 300 g were implanted with an indwelling intrathecal catheter (PE-10; 0.75 mm diameter) under fluothane anesthesia using a modification of the method described by Yaksh and Rudy (1976). The distal end of the catheter was inserted through the atlanto-occipital membrane into the spinal cord subarachnoid space and threaded to the rostral edge of the lumbar enlargement (8.5 cm). The proximal end of the catheter was externalized through an incision of the skin 1 cm anterior to the occipital ridge. Animals were housed individually after surgery with free access to food and water. All analgesiometric testing was performed on the seventh postoperative day. Those animals which displayed motor deficits were discarded from the study.

2.2. A nalgesiometric testing Two analgesiometric tests were utilized in this study: the tail flick test (D'Amour and Smith, 1941) and the hot plate test (Woolfe and MacDonald, 1944). The tail flick test was conducted by positioning the rat's tail over a slit in a metal plate

through which the light of a 300 W quartz bulb was focused. The time which elapsed from onset of the thermal stimulus to reflex removal of the tail (flick) from the light source was termed the tail flick latency (TFL). In order to prevent tissue damage, the trial was terminated after 10 s had elapsed. The mean of two trials was recorded. The hot plate test was performed by placing the animal on a metal surface heated to 52.5°C by a temperature regulated water bath. The time which elapsed from placement of the animal on the hot plate to a lick of the hind paw was termed the hot plate latency (HPL). Cut-off time on the hot-plate test was 60 s.

2.3. Administration of drugs A hand-driven syringe pump was used for intrathecal injection. Each drug was microinjected in a volume of 15 /~1 and the catheter was then flushed with 10/~1 saline to ensure delivery of the drug to the caudal intrathecal space. Clorgyline, the only drug administered systemically, was injected intramuscularly in a volume of 0.1 ml/100 g body weight 2 h prior to analgesiometric testing.

2.4. Experimental procedure Two hours after the i.m. administration of clorgyline (5 mg/kg), an inhibitor of monoamine oxidase (Johnston, 1968), baseline T F L and HPL were determined for all rats. Each rat was then injected intrathecally with either a 5-HT antagonist or with saline vehicle (pH 7.0). Ten min later T F L and HPL were measured to determine whether the injection of saline vehicle or the 5-HT antagonists had altered nociceptive threshold. Either 100 or 200 /Lg 5-HT creatinine sulfate was injected intrathecally immediately afterward. Tail flick and hot plate latencies were measured 5, 15 and 25 min later. Each rat received only one dose of antagonist and agonist.

2.5. Drugs The following drugs were used in the study: 5-hydroxytryptamine creatinine sulfate (Regis), methysergide maleate (Sandoz), metergoline

351 (Farmitalia), ketanserin (3-(2-[4-(fluorobenzoyl)1-piperdinyl] ethyl)-2,4-[ 1H,3H] quinazolinedione, Janssen), spiroperidol (Janssen), 2-bromolysergic acid diethylamide tartrate (NIDA), phentolamine hydrochloride (Ciba-Geigy), clorgyline (May & Baker), and creatinine sulfate (Sigma). Doses of all drugs were calculated as the salt, put into solution using physiological saline on the day of use, and the pH adjusted to as near 7.0 as possible. Solutions of 5-HT and creatinine sulfate were allowed to remain at pH 3.0.

2.6. Distribution of [14C]5-HT in the CNS The distribution of [~4C]5-HT in the CNS following its intrathecal administration was examined in a supplemental study. Seven days after implantation of an intrathecal catheter, male SpragueDawley rats received 5 m g / k g clorgyline i.m. Two hours later, 15 /~1 of a solution which contained 200 /xg 5-HT creatinine sulfate and 0.05 /~Ci of [14C]5-HT creatinine sulfate (Amersham; 53 m C i / m m o l ) was injected intrathecally followed by a 10 /~1 flush with saline. Mean counts per min administered intrathecally was 149018.7 + 3 141.4 ( + S.E.). Two rats were each killed by decapitation 5, 15 and 45 min after injection. Samples of diencephalon, brain stem and lumbar spinal cord were rapidly removed and frozen on dry ice. Tissue samples were solubilized using 0.7 ml of Soluene 350. Seventy-two hours later, the radioactivity of the samples was determined using a Beckmann liquid scintillation spectrometer (model LS 8800) with a mean counting efficiency of 80.7 + 0.1% (___S.E.). All tissue counts were normalized to 100 mg tissue wet weight.

man-Keuls test for multiple comparisons (Keppel, 1973). Examination of the time course of the effect of intrathecally administered 5-HT on nociceptive threshold (see 3.1.) indicated that ~there was no significant difference between the response latency measured 5 and 15 min after injection of 5-HT. Thus, the maximum response latency measured in the 15 min following injection of 5-HT was used to construct the dose-response curves. One-way analyses of variance were used to compare the alteration in response latencies produced by intrathecal injection of 5-HT creatinine sulfate with those produced by intrathecal injection of creatinine sulfate. Newman-Keuls test was used to make comparisons between the individual doses of 5-HT. The response latencies of the individual animals were used to estimate the line of best fit by the least-squares method of linear regression. In that portion of the study which examined the ability of serotonergic antagonists to antagonize the increase in T F L and HPL produced by 5-HT, the maximum response latencies measured in the 15 min following injection of 5-HT were used to construct the dose-response curves for the antagonists. One-way analyses of variance in conjunction with Newman-Keuls test for multiple comparisons were used to determine the significance of the antagonists' effects on the elevation of nociceptive threshold produced by 5-HT. The line of best fit for the response latencies of the individual animals after each antagonist was estimated using the least squares method of linear regression. The IDs0 of the antagonists and confidence limits were determined according to Tallarida and Murray (1981).

2. 7. Statistical analysis 3. Results

In that portion of the study which examined the effect of the serotonergic antagonists on nociceptive threshold, the difference (zx) between the baseline response latency and the response latency measured 10 min after intrathecal injection of the antagonists was determined. These values were then compared to the zx produced by intrathecal injection of saline by means of a one-way analysis of variance (ANOVA) in conjunction with New-

3.1. Effect of mtrathecally administered 5-HT on nociceptive threshold Intrathecal administration of 5-HT creatinine sulfate significantly elevated nociceptive threshold as measured by the tail flick and hot plate tests in both untreated rats and in rats pretreated with clorgyline, an inhibitor of monoamine oxidase

352

(Johnston, 1968). The maximum T F L produced by intrathecal injection of 200 ttg 5-HT was the same in untreated rats (6.7 + 0.5 s; n = 10) and clorgyline-treated rats (7.4 + 0.4 s; n = 14) (ANOVA, P > 0.05). However, the maximum HPL produced by this dose of 5-HT was greater in clorgylinetreated rats (60.0+ 0.0 s; n = 14) than in untreated rats (54.0 + 3.5 s; n = 10) (Mann-Whitney U-test, P < 0.05). In addition, the elevation of T F L and HPL was more consistently apparent and of longer duration in the clorgyline-treated rats. Similar effects were observed with 100 /~g 5-HT. For this reason, all subsequent experiments were performed using rats which had been pretreated with 5 m g / k g clorgyline 2 h prior to analgesiometric testing. The elevation of T F L produced by intrathecal administration of 5-HT was rapid in onset and of short duration. The elevation of mean T F L was maximal at 5, remained unchanged at 15 min after injection of 5-HT, and had returned to baseline values by 35 min after injection. Fig. 1 illustrates that the magnitude of the elevation of T F L produced by intrathecal injection of 5-HT creatinine sulfate was dose dependent. Both 100 and 200/~g 5-HT significantly elevated T F L (P < 0.05) as compared to T F L after intrathecal injection of 109 ~g creatinine sulfate, a dose equimolar to the 200 ~g dose of 5-HT. Fifty ktg 5-HT creatinine sulfate did not significantly elevate TFL. As observed with the tail flick test, the elevation of HPL produced by intrathecally administered 5-HT was also maximal 5 and 15 rain after its injection. However, the duration of the elevation of HPL exceeded that of T F L and was still evident 35 rain after injection of the 5-HT. The elevation of HPL was dose dependent and produced by injection of as little as 50 t~g 5-HT (fig. 1). Administration of clorgyline might be expected to enhance the actions of endogenous norepinephrine in the spinal cord. Since intrathecal injection of noradrenergic agonists has been shown to elevate nociceptive threshold (Kuraishi et al., 1979; Reddy et al., 1980), it was important to determine the extent to which the blockade of norepinephrine metabolism might have contributed to the elevation of T F L and H P L observed following intrathecal administration of 5-HT in clorgyline-treated

8

TAIL

FLICK

7

>',..) Z W

6 5

I

60-

HOT

I

I

I

l

I

I I 50

I

I II

I

I

PLATE

504030-

f

Io

I

DOSE

I

II

I

I

I00 5-HT

200

(.ug)

Fig. I, Elevation of nociceptive threshold following intrathecal administration of 5-hydroxytryptamine creatinine sulfate as measured using the tail flick (top) and hot plate (bottom) tests. Ordinate: response latency in seconds. Abscissa: log dose of 5-HT in Fg. x Indicates significant difference as compared to mean response latency following intrathecal administration of 109/Lg creatinine sulfate (11) (ANOVA, P < 0.05). Solid triangle (A) indicates mean response latency following intrathecal injection of 100/~g 5-HT in rats injected intrathecally 10 rain earlier with 48 nmol phentolamine hydrochloride. Lines of best fit were estimated by the least squares method of linear regression using individual values for all three doses on the tail flick test and for the two lowest doses on the hot plate test. The r values for the tail flick and hot plate tests were 0.64 and 0.55, respectively (P < 0.05; each line). Each point represents the mean + S.E. of determinations made in 10-14 rats. The mean TFL and HPL measured 10 min after the intrathecal injection of saline in these 34 rats were 4.0_+0.1 and 21.7_+1.7 s, respectively.

animals. However, intrathecal injection of 48 nmol of phentolamine hydrochloride, a dose previously demonstrated to effectively antagonize the elevation of T F L produced by intrathecally injected norepinephrine (Reddy et al., 1980), failed to antagonize the elevation of T F L and HPL produced by intrathecal injection of 100/~g 5-HT (fig. 1). Thus, norepinephrine does not appear to contribute to the ability of intrathecally administered 5-HT to elevate T F L and HPL in clorgyline-treated rats.

353

3.2. Effects of intrathecally administered serotonergic antagonists on nociceptive threshold A o n e - w a y analysis of variance was used to c o m p a r e the alteration in nociceptive threshold p r o d u c e d b y intrathecal a d m i n i s t r a t i o n of the serotonergic antagonists with that produced by intrathecal a d m i n i s t r a t i o n of saline. T e n m i n after intrathecal injection of saline in 34 rats, m e a n T F L was 4.0 + 0.1 s a n d m e a n H P L was 21.7 + 1.7 s, differences of 0.4 4- 0.1 a n d 4.0 4. 1.4 s, respectively, from preinjection baseline latencies. At the

-j

3.3. Effects of intrathecally administered serotonergic antagonists on the 5-HT-induced elevation of nociceptive threshoM

~J

z ku

y_

,5-

J LL

4-

highest doses used in this study, neither methysergide (48 nmol), metergoline (32 nmol), 2bromolysergic acid diethylamide tartrate (BOL) (48 nmol), n o r ketanserin (48 n m o l ) significantly altered either T F L or H P L 10 m i n after their a d m i n i s t r a t i o n as compared to saline. The highest dose of spiroperidol (44 nmol), however, produced a slight decrease in T F L (by 0.5 + 0.1 s; n = 10) which was significant when compared to the slight increase in T F L (by 0.5 + 0.1 s; n = 10) produced b y saline. Spiroperidol did n o t alter H P L as compared to saline.

~-3I 5

DOSE

I 1o

ANTAGONIST

I ~o

I 30

I r 40 ~o

I

1oo

(nmoles)

Fig. 2. Effect of intrathecally administered methysergide (A), BOL (I), spiroperidol (zx), ketanserin (I) and metergoline (©) on the elevation of TFL produced by intrathecal administration of 200 #g 5-HT creatinine sulfate. Ordinate: tail flick latency in seconds. Abscissa: log dose of the antagonist in nmol. * Indicates significant difference from mean TFL followingintrathecal injection of 200 /~g 5-HT in rats injected intrathecally 10 min earlier with saline. Lines of best fit were calculated using individual values and the least squares method for linear regression. Those doses in which the effect of the antagonist had reached an asymptote were not included in the analysis. The r values for methysergide, BOL and ketanserin were -0.55, -0.40 and -0.58, respectively (P<0.05; each drug). The correlation coefficients for metergoline (-0.23) and spiroperidol (-0.19) were not significant (P > 0.05; both drugs). Each point represents the mean ±S.E. of determination made in 8-10 rats. At least three doses of an antagonist were examined, but for reasons of clarity the following doses which fell on either asymptote of their curve, were omitted from the graph: BOL, 6 nmol; methysergide, 48 nmoles; ketanserin, 12 and 73 nmol; spiroperidol, 6 nmol. Intrathecal injection of 200 #g 5-HT creatinine sulfate increased TFL to 7.4+0.4 s (95% confidence limits: 6.6-8.3 s). Intrathecal injection of saline resulted in a mean TFL of 4.0+0.1 s (n = 34; 95% confidence limits 3.8-4.2 s).

Methysergide, BOL, spiroperidol a n d ketanserin each antagonized the elevation of T F L produced by intrathecal a d m i n i s t r a t i o n of 100 or 200 /zg 5 - H T creatinine sulfate in a dose d e p e n d e n t m a n n e r ( A N O V A , P < 0.05; each c o m p o u n d ) (fig. 2). Of these four antagonists, methysergide was the most potent, with a m e a n I£)50 of 17.5 + 2.5 nmol. K e t a n s e r i n was the least p o t e n t antagonist, with a m e a n IDs0 of 34.5 + 5.1 nmol. The slopes of the linear regression lines d r a w n for methysergide, BOL and k e t a n s e r i n were not significantly different ( P > 0.05). A l t h o u g h the highest dose of metergoline (32 n m o l ) used in this study att e n u a t e d the 5 - H T i n d u c e d increase in T F L , this a n t a g o n i s m was n o t dose d e p e n d e n t between 6 a n d 32 n m o l ( A N O V A , P > 0.05) (fig. 2). T a b l e 1 lists the ID~o S.E. a n d confidence limits de-

TABLE 1 IDs0 and confidence limits determined for the effect of intrathecally administered 5-HT antagonists on the 5-HT-induced elevation of TFL in the rat ~. Antagonist

IDs0 ~

S.E.

Confidence b limits

Methysergide BOL Ketanserin

17.5 26.5 34.5

2.5 6.5 5.1

12.2-20.0 13.5-39.5 24.8-45.2

a iDso and confidence limits were determined according to Tallarida and Murray (1981). b nmol.

354

termined for methysergide, BOL and ketanserin. As the slope of the linear regression lines drawn for metergoline and spiroperidol were not significant, an IDs0 was not determined for these antagonists. In marked contrast to the results obtained with the tail flick test, none of the antagonists examined in this study attenuated the elevation of HPL produced by intrathecal administration of either 100 or 200 #g 5-HT (Kruskall-Wallis, P > 0.05; each compound) (data not shown).

3.4. Behavioural observations Approximately 25 min after the intrathecal injection of 200 #g 5-HT in a clorgyline-treated rat a complex behavioral syndrome consisting of hyperreactivity, resting tremor, rigidity or hypertonus, mild hindlimb abduction and pronounced salivation was observed. This syndrome has been described previously as a behavioral index of central serotonergic activity (Jacobs and Klemfuss, 1975). The peak effect was seen 45 min postinjection. These behavioral signs appeared more pronounced in experiments where phentolamine and spiroperidol were intrathecally administered. These animals additionally showed more pronounced myoclonic signs, as well as head shaking, extensive agitation and jumping. 3.5. Distribution of intrathecally administered [14C]5-HT

The distribution of [~4C]5-HT in brain and spinal cord at various times after its intrathecal injection is presented in table 2. It is apparent that 5 and 15 rain after its injection, a substantial portion of the intrathecally administered [~4C]5H T remained in the lumbar spinal cord. In comparison, a very small amount had distributed to either the brain stem or diencephalon. Forty-five rain after injection, however, the amount ot [14C]5-HT in the spinal cord had decreased while that in the brain stem and diencephalon had increased. The fact that the rats were pretreated with clorgyline increased the likelihood that the radioactivity was associated with the 5-HT molecule rather than monoamine oxidase byproducts.

TABLE 2 Distribution in the CNS of intrathecally administered [14C]5H T creatinine sulfate. Time a

Tissue Lumbar spinM cord

Brainstem

Diencephalon

5 15 45

5 300, 14310 b 7260, 4440 3740, 909.3

58, 7 155,321 266, 70

33, 4 36,88 270, 63

Minutes after intrathecal injection of 200/~g 5-HT creatinine sulfate and 0.05 ttCi of [~4C]5-HT creatinine sulfate; 181 868 + 4067 d p m (mean + S.E.). b Expressed as d p m / 1 0 0 mg tissue wet weight; n = 2 at each time point.

4. Discussion

As reported in an earlier study (Yaksh and Wilson, 1979), intrathecal administration of 5-HT creatinine sulfate produced a significant elevation of nociceptive threshold as measured using two different analgesiometric tests, the tail flick and the hot plate. Creatinine sulfate alone in equimolar doses had no effect. The magnitude of the elevation of T F L and HPL was dose dependent. It was also rapid in onset, appearing within 5-15 min of injection. The failure to see correspondence between the time course of analgesia and the peak concentrations of [tac]5-HT in brain following spinal administration supports the argument that the elevation of nociceptive threshold measured after intrathecally administered 5-HT is the result of an action of 5-HT exerted at the level of the spinal cord. A similar restriction of labeled compounds to the spinal cord has been reported by other investigators following the intrathecal administration of a variety of radiolabeled agents (Yaksh and Rudy, 1976; Lopachin and Rudy, 1982). Significantly, the time course of the behavioral side effects observed following the intrathecal administration of 5-HT (hyperreactivity, resting tremor and salivation), thought to be mediated by supraspinal serotonergic mechanisms (Jacobs and Klemfuss, 1975), correlates well with the time course of the supraspinal redistribution of intrathecally administered [ 14C]5-HT. In this study, the effects of intrathecally admin-

355 istered 5-HT and 5-HT antagonists on nociceptive threshold were assessed in rats pretreated with clorgyline, an inhibitor of monoanaine oxidase. In agreement with a previous report (Yaksh and Wilson, 1979), pretreatment of the rats with an inhibitor of monoamine oxidase potentiated the action of 5-HT. This potentiation cannot be attributed to a concomitant elevation of norepinephrine content in the spinal cord since intrathecal injection of the noradrenergic antagonist phentolamine did not attenuate the elevation of T F L or HPL produced by intrathecally administered 5-HT in clorgylinetreated rats. It has been suggested that the two proposed 5-HT receptor populations may have different physiological functions (Peroutka et al., 1981). With regard to the pharmacological nature of the spinal receptor related to nociception, methysergide, metergoline, BOL, ketanserin and spiroperidol were examined for their ability to antagonize the 5-HT-induced elevation of T F L and HPL when administered intrathecally. Methysergide, BOL and metergoline bind to both 5-HT t and 5-HT2 receptor sites. In contrast, ketanserin and spiroperidol show a relatively selective affinity for the 5-HT2 receptor alone (Leysen et al., 1981). In the present experiments, methysergide was the most potent antagonist, followed by BOL with intermediate potency. Metergoline attenuated the T F L at the highest dose which was soluble. In contrast, ketanserin was the least potent compound with an IDs0 significantly greater than that of methysergide. Although spiroperidol, frequently used to characterize the 5-HT2 receptor (Creese and Snyder, 1978; Peroutka and Snyder, 1979; 1981), attenuated the elevation of T F L produced by 5-HT, this antagonism occured only at a dose which alone produced significant decrease in TFL. It thus does not appear that activation of 5-HT 2 receptors in the spinal cord contributes measurably to the production of analgesia by 5-HT as measured using the tail flick test, since both spiroperidol and ketanserin were the least potent antagonists. This interpretation is supported by the recent report of Blackshear and coworkers (1981) and Monroe and Smith (1982) that the 5-HT~ receptor is the predominant species in the spinal cord.

The present systematic study of the effects of intrathecally administered methysergide, metergoline, BOL, ketanserin and spiroperidol failed to show any antagonism of the effects of intrathecally administered 5-HT on the HPL. Since the justmaximal elevation of HPL observed following injection of 100/~g 5-HT was also not affected by any of these compounds, the choise of an inappropriately low cutoff latency cannot be the reason for failure to see antagonism. As the present series of studies were carried out over a wide range of doses with a variety of agents, we do not believe the question of adequate drug levels in the spinal cord apply. These results differ from previous findings in which systemically administered methysergide or cyproheptadine were observed to produce a dose dependent reduction in the effects of 5-HT on both T F L and HPL (Yaksh and Wilson, 1978). Two factors distinguish this study from the previous study. First, in the previous study, agitation/repeated lifting of the hind paws o r a lick of the hind paw were considered as the endpoint response on the hot plate test, whereas only a lick of the hind paw was accepted in the present study. Thus, the results of the original study (Yaksh and Wilson, 1979) in which the hot plate results were interpreted as a supraspinally mediated response may have been confounded by a strong spinal reflexive motor component since repeated lifting of the hind paw was also accepted as an endpoint response. Second, in the present study, all rats were pretreated with clorgyline which potentiated the action of 5-HT in the spinal cord. The disparate results obtained with the tail flick and hot plate tests in this study illustrate a problem inherent to behavioral tests of nociception, the involvement of a motor component in the endpoint response. Although both the tail flick and hot plate tests have been used successfully as measures of 'analgesia', they do differ and may be differentially affected by drugs. Thus, the tail flick test has a motor component whose contribution to the final response measure cannot be directly assessed. In contrast, the hot plate test involves supraspinally organized neural circuitry which provides for a coordinated motor activity secondary to the arrival of the appropriate stimulus information at supraspinal structures. Failure to

356

see normal elements of this coordinated motor behavior (i.e. ambulation, grooming, etc.) provides clear evidence for disruption of motor activity that could lead to erroneous interpretations of the analgesic efficacy of the agent. In summary, the results of the present study confirm earlier reports (Wang, 1977; Yaksh and Wilson, 1979) that intrathecal administration of 5-HT produces an elevation of nociceptive threshold. In addition, the pharmacological profile of the antagonists in this study leads to the suggestion that activation of a 5-HT~ receptor, rather than of a 5-HT2 receptor may be the predominant, though not exclusive, mechanism by which intrathecally administered 5-HT alters spinal nociceptive reflexes. Definitive support for this postulate, however, must await the synthesis of antagonists having a selective affinity for 5-HT~ receptors.

Acknowledgements This work was supported by PHS grant NS 16541 to TLY and Postdoctoral Fellowship NS 06538 to DLH. We thank Ms. Gail Harty for her technical assistance and Ms. Ann Rockafellow for preparation of the manuscript.

References Blackshear, M.A., L.R. Steranka and E. Sanders-Bush, 1981, Multiple serotonin receptors: regional distribution and effect of raphe lesions, European J. Pharmacol. 76, 325. Creese, I. and S.H. Snyder, 1978, 3H-Spiroperidol labels serotonin receptors in rat cerebral cortex and hippocampus, European J. Pharmacol. 49, 201. D'Amour, F.E. and D.L. Smith, 1941, A method for determining loss of pain sensation, J. Pharmacol. Exp. Ther. 72, 74. Griersmith, B.T. and A.W. Duggan, 1978, Prolonged depression of spinal transmission of nociceptive information by 5-HT administered in the substantia gelatinosa: antagonism by methysergide, Brain Res. 187, 231. Haigler, H.J. and G.K. Aghajanian, 1974, Peripheral serotonin antagonists: failure to antagonize serotonin in brain areas receiving a prominent serotonergic input, J. Neurol. Trans. 35, 257. Haigler, H.J. and G.K. Aghajanian, 1977, Serotonin receptors in the brain, Fed. Proc. 36, 2159. Hamon, M., D.L. Nelson, A. tlerbet and J. Glowinski, 1980, Multiple receptors for serotonin in the rat brain, in: Receptors for Neurotransmitters and Peptide Hormones, eds. G.

Pepeu, M.J. Kuhar and S.J. Enna (Raven Press, New York) p. 223. Headley, P.M., A.W. Duggan and B.T. Griersmith, 1978, Selective reduction by noradrenaline and 5-hydroxytryptamine of nociceptive responses of cat dorsal horn neurones, Brain Res. 145, 185. Jacobs, B.L. and H. Klemfuss, 1975, Brain stem and spinal cord mediation of a serotonergic behavioral syndrome, Brain Res. 100, 456. Johnston, J.P., 1968, Some observations upon a new inhibitor of monoamine oxidase in brain tissue, Biochem. Pharmacol. 17, 1285. Jordan, L.M., D.R. Kenshalo, Jr., R.F. Martin, L.H. Haber and W.D. Willis, 1978, Depression of primate spinothalamic tract neurons by iontophoretic application of 5-hydroxytryptamine, Pain 5, 135. Keppel, G., 1973, Design and Analysis: A Researcher's Handbook (Prentice-Hall, New Jersey). Kuraishi, Y., Y. Harada and H. Takagi, 1979, Noradrenaline regulation of pain transmission in the spinal cord mediated by a-adrenoceptors, Brain Res. 174, 333. Leysen, J.E., F. Awouters, L. Kennis, P.M. Laduron, J. Vandenberk and P.A.J. Janssen, 1981, Receptor binding profile of R41 468, a novel antagonist at 5-HT2 receptors, Life Sci. 28, 1015. Lopachin, R.M. and T.A. Rudy, 1982, Sites and mechanism of action for the effects of intrathecal norepinephrine on thermoregulation, J. Physiol. (London) (in press). McCall, R.B. and G.K. Aghajanian, 1980, Pharmacological characterization of serotonin receptors in the facial motor nucleus: a microiontophoretic study, European J. Pharmacol. 65, 175. Messing, R.B. and L. Lytle, 1977, Serotonin-containing neurons: their possible role in pain and analgesia, Pain 4, 1. Monroe, P.J. and D.J. Smith, 1982, Characterization of multiple [3H]5-hydroxytryptamine binding sites in rat spinal cord, Neurosci. Abst. 8, 647. Peroutka, S.J., R.M. Lebovitz and S.H. Snyder, 1981, Two distinct central serotonin receptors with different physiological functions, Science 212, 827. Peroutka, S.J. and S.H. Snyder, 1979, Multiple serotonin receptors: differential binding of [3H]5-hydroxytryptamine [3H]lysergic acid diethylamide and [3H]spiroperidol, Mol. Pharmacol. 16, 687. Peroutka, S.J. and S.H. Snyder, 1981, Two distinct serotonin receptors: regional variations in receptor binding in mammalian brain, Brain Res. 208, 339. Randic, M. and H.H. Yu, 1976, Effects of 5-hydroxytryptamine and bradykinin in cat dorsal horn neurones activated by noxious stimuli, Brain Res. 111, 197. Reddy, S.V.R. and T.L. Yaksh, 1980, Spinal noradrenergic terminal system mediates antinociception, Brain Res. 189, 391. Segal, M., 1976, 5-HT antagonists in rat hippocampus, Brain Res. 103, 161. Tallarida, R.J. and R.B. Murray, 1981, Manual of Pharmacologic Calculations with Computer Programs (SpringerVerlag, New York).

357 Wang, J.K., 1977, Antinociceptive effect of intrathecally administered serotonin, Anesthesiology 47, 269. Woolfe, G. and A.D. MacDonald, 1944, The evaluation of the analgesic action of pethidine hydrochloride (Demerol), J. Pharmacol. Exp. Ther. 80, 300.

Yaksh, T.L. and T.A. Rudy, 1976, Chronic catheterization of the spinal subarachnoid space, Physiol. Behav. 17, 1031. Yaksh, T.L. and P.R. Wilson, 1979, Spinal serotonin terminal system mediates antinociception, J. Pharmacol. Exp. Ther. 208, 446.